Adenylyl cyclase is the enzyme that converts ATP to cyclic AMP (cAMP). cAMP is an intracellular second messenger that controls diverse physiological responses. Several human pathogens secrete highly active adenylyl cyclase toxins. Anthrax bacteria secrete edema factor (EF) while pertussis bacteria, which cause whooping cough, secrete CyaA. Both EF and CyaA can elevate intracellular cAMP to pathologic levels, altering the human physiology and immune responses. EF and CyaA share a homologous adenylyl cyclase domain (ACD, 25% similarity) and both toxins become active when they enter host cells and bind a calcium sensor, calmodulin (CaM). In the previous funding period of this grant, we used the structural and biochemical analyses to contruct a working model of how CaM binds and activates EF, and how EFperforms the cyclization reaction. We have also solved the structure of CyaA-ACD in complex with the C-terminal domain of CaM (C-CaM). Surprisingly, while CyaA-ACD is structurally similar to EF-ACD, the interaction of CyaA-ACD with C-CaM diverges completely from that of EF-ACD with C-CaM. Our current understanding in how CaM binds and activates CyaA-ACD is incomplete. I propose to perform biochemical and structural analyses to address how CaM binds and activates CyaA-ACD. In addition, I propose to use structural and computational analyses to address how EF and CyaA conduct their cyclization reaction. Success in these aims will provide a better understanding of how these two structurally related toxins evolve divergently to bind an evolutionary conserved calcium sensor, CaM. Success in these aims will also shed light on how CaM could interact with its targets in eukaryotic cell signaling. In addition, this will provide valuable information in designing drugs to prevent the pathologic consequences resulting from bioterrorism and emerging infections. EF requires an association with anthrax protective antigen (PA) to enter host cells. We also propose to use surface plasmon resonance sensorgram and structural analyses to address how EF binds PA. An improved understanding of the interaction between EF and PA should provide a better means of designing inhibitors to prevent EF from entering host cells.